Class II major histocompatibility complex (MHC-II) proteins play a central role in the control of normal immune homeostasis, while aberrant expression of MHC-II is frequently associated with abnormalities in immune responses. MHC-II proteins elicit immune activation through presentation of exogenously derived antigens to CD4 ϩ T cells and represent the seminal control of both peripheral T-cell activation and thymic selection (23,28,47). The level of MHC-II expression is exquisitely regulated. Constitutive MHC-II expression is restricted to B cells, monocytes, macrophages, and dendritic cells, whereas inducible expression is observed on a selected number of cell types in response to cytokines such as gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) (37, 47). The regulation of MHC-II expression resides predominantly at the transcriptional level and is globally controlled by the master regulator, class II transactivator (CIITA) (12, 47).CIITA was initially isolated by complementation cloning, using an Epstein-Barr virus-based library to rescue MHC-II expression in MHC-II-negative cells (45). CIITA is encoded by the MHC2TA gene, deletions in which represent the genetic defect in immunodeficient type II group A bare lymphocyte syndrome patients. Expression of CIITA is controlled by four distinct promoters, allowing for a complex pattern of constitutive and inducible MHC-II expression (31, 39). CIITA does not bind DNA but controls MHC-II and related genes by interacting with the requisite MHC-II transcription factors (RFX5, CREB, and NF-Y), which associate with conserved promoter motifs, termed X1, X2, and Y, respectively (9,26,29,42,58). These interactions are critical for the formation of a stable enhanceosome. CIITA also interacts with components of the basal transcription machinery (TFIIB, TATA binding protein, and TATA binding protein-associated factors) (6,25,27). Most relevant to this work, CIITA associates with several chromatin remodeling enzymes, including histone acetyltransferases (HATs) CBP/p300, and pCAF (16,43,44,59), and ATPdependent remodeling factors, such as BRG-1 (30, 38). These enzymes have all been demonstrated to modulate MHC-II promoter activation.Structure-function analysis of CIITA protein indicates that it can be divided into three important segments. The N terminus contains an acidic transactivation domain as well as target lysines for both acetylases and a HAT-like domain (16,40,44). The mid-section contains a nucleotide-binding domain (NBD) that is critical for nuclear import and contributes to self-association (10,17,21). The C terminus contains a stretch of leucine-rich repeats (LRRs) that are also involved in proteinprotein association (11,21). This unique combination of the NBD and LRR domains is a conserved feature among a new family of known and novel genes, which we have recently called the CATERPILLER family (11). The NBD domain is also shared by a more loosely related family of known genes, called the NACHT family. Members of this family range from plant

Although increasing evidence indicates that there is a direct link between ubiquitination and mono-ubiquitination and transcription in yeast, this link has not been demonstrated in higher eukaryotes. Here we show that the major histocompatibility complex (MHC) class II transactivator (CIITA), which is required for expression of genes encoding MHC class II molecules, is ubiquitinated. This ubiquitination enhanced the association of CIITA with both MHC class II transcription factors and the MHC class II promoter, resulting in an increase in transactivation function and in the expression of MHC class II mRNA. The degree of CIITA ubiquitination was controlled by histone acetylases (HATs) and deacetylases (HDACs), indicating that the crucial cellular processes mediated by these enzymes are linked to regulate transcription. Thus, ubiquitin positively regulates a mammalian coactivator by enhancing its assembly at the promoter.

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405–414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

RGS10 is an important regulator of cell survival and chemoresistance in ovarian cancer. We recently showed that RGS10 transcript expression is suppressed during acquired chemoresistance in ovarian cancer. The suppression of RGS10 is due to DNA hypermethylation and histone deacetylation, two important mechanisms that contribute to silencing of tumor suppressor genes during cancer progression. Here, we fully investigate the molecular mechanisms of epigenetic silencing of RGS10 expression in chemoresistant A2780-AD ovarian cancer cells. We identify two important epigenetic regulators, HDAC1 and DNMT1, that exhibit aberrant association with RGS10 promoters in chemoresistant ovarian cancer cells. Knockdown of HDAC1 or DNMT1 expression, and pharmacological inhibition of DNMT or HDAC enzymatic activity, significantly increases RGS10 expression and cisplatin-mediated cell death. Finally, DNMT1 knock down also decreases HDAC1 binding to the RGS10 promoter in chemoresistant cells, suggesting HDAC1 recruitment to RGS10 promoters requires DNMT1 activity. Our results suggest that HDAC1 and DNMT1 contribute to the suppression of RGS10 during acquired chemoresistance and support inhibition of HDAC1 and DNMT1 as an adjuvant therapeutic approach to overcome ovarian cancer chemoresistance.

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Chromatin immunoprecipitation (ChIP) assays were developed in order to comprehensively describe physiological interactions between DNA sequences, transcriptional regulators, and the modification status of associated chromatin. In ChIP assays, living cells are treated with chemical cross-linkers to covalently bind proteins to each other and to their DNA targets. Once cross-linked to associated proteins, chromatin is extracted and fragmented by sonication and protein-DNA complexes are isolated using specific antibodies against a target protein. The cross-links that bind proteins to DNA are then reversed, and purified DNA fragments are analyzed by qPCR to determine if a specific sequence is present. As DNA regulatory elements frequently rely on the interaction of multiple transcription factors and cofactors to regulate gene expression, Re-ChIP methods were developed to allow for the identification of multiple (concurrently binding) proteins on a single DNA sequence. Re-ChIP assays have enabled the analysis of multiple, simultaneous, posttranslational modifications to histones in order to determine the combinatorial pattern of modifications associated with transcriptional status of a gene. Together, ChIP and Re-ChIP have contributed to the elucidation of the epigenetic code-regulating gene expression and have enhanced our understanding of physiological binding of proteins to DNA targets. The protocols that follow describe general strategies used to perform ChIP and Re-ChIP assays for the study of specific protein-DNA interactions.

Recent studies have made evident the fact that the 19S regulatory component of the proteasome has functions that extend beyond degradation, particularly in the regulation of transcription. Although 19S ATPases facilitate chromatin remodeling and acetylation events in yeast (Saccharomyces cerevisiae), it is unclear if they play similar roles in mammalian cells. We have recently shown that the 19S ATPase Sug1 positively regulates the transcription of the critical inflammatory gene for major histocompatibility complex class II (MHC-II) by stabilizing enhanceosome assembly at the proximal promoter. We now show that Sug1 is crucial for regulating histone H3 acetylation at the MHC-II proximal promoter. Sug1 binds to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at the proximal promoter, with a preferential loss of acetylation at H3 lysine 18. Sug1 also binds to the MHC-II histone acetyltransferase CREB-binding protein (CBP) and is critical for the recruitment of CBP to the MHC-II proximal promoter. Our current study strongly implicates the 19S ATPase Sug1 in modifying histones to initiate MHC-II transcription and provides novel insights into the role of the proteasome in the regulation of mammalian transcription.Major histocompatibility complex class II (MHC-II) molecules are cell surface glycoproteins which bind and present processed antigenic peptides to CD4ϩ T lymphocytes to initiate immune system protection against invading pathogens and tumors (57). Tight regulation of MHC-II expression is crucial to maintain a functional immune system and to limit the opportunity for the development of autoimmune diseases (32, 57). MHC-II is expressed constitutively on antigen-presenting cells and can be inducibly expressed on most nucleated cells by gamma interferon (IFN-␥) (7,20). Constitutive and IFN-␥-inducible MHC-II expression is regulated at the level of transcription by a series of elements in the MHC-II promoter. Nuclear factor Y, regulatory factor X, and cyclic AMP response element binding protein (CREB) bind, respectively, to the Y and X elements of the MHC-II proximal promoter, forming a multiprotein enhanceosome complex, which is necessary but not sufficient for transcription initiation. Once the enhanceosome is assembled on the MHC-II promoter, the class II transactivator (CIITA) can be recruited. CIITA binding stabilizes the enhanceosome complex and recruits basal transcriptional components, including the CDK7 subunit of TFIIH and the CDK9 subunit of P-TEFb, which phosphorylate polymerase II and initiate the switch to an elongation complex (12,41,48,50,68). CIITA is also known to interact with a variety of transcriptional cofactors, including multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) (8,29,76,81). Although much is known about the requirement of these basal and inducible transcription factors for MHC-II expression, less is known regarding the importance of epigenetic modifications required to open the chromatin structure and all...

RGS10 regulates ovarian cancer cell growth and survival, and RGS10 expression is suppressed in cell models of ovarian cancer chemoresistance. However, the mechanisms governing RGS10 expression in ovarian cancer are poorly understood. Here we report RGS10 suppression in primary ovarian cancer and CAOV-3 ovarian cancer cells compared to immortalized ovarian surface epithelial (IOSE) cells, and in A2780-AD chemoresistant cells compared to parental A2780 cells. RGS10-1 and RGS10-2 transcripts are expressed in ovarian cancer cells, but only RGS10-1 is suppressed in A2780-AD and CAOV-3 cells, and the RGS10-1 promoter is uniquely enriched in CpG dinucleotides. Pharmacological inhibition of DNA methyl-transferases (DNMTs) increased RGS10 expression, suggesting potential regulation by DNA methylation. Bisulfite sequencing analysis identified a region of the RGS10-1 promoter with significantly enhanced DNA methylation in chemoresistant A2780-AD cells relative to parental A2780 cells. DNA methylation in CAOV-3 and IOSE cells was similar to A2780 cells. More marked differences were observed in histone acetylation of the RGS10-1 promoter. Acetylated histone H3 associated with the RGS10-1 promoter was significantly lower in A2780-AD cells compared to parental cells, with a corresponding increase in histone deacetylase (HDAC) enzyme association. Similarly, acetylated histone levels at the RGS10-1 promoter were markedly lower in CAOV-3 cells compared to IOSE cells, and HDAC1 binding was doubled in CAOV-3 cells. Finally, we show that pharmacological inhibition of DNMT or HDAC enzymes in chemoresistant A2780-AD cells increases RGS10 expression and enhances cisplatin toxicity. These data suggest that histone de-acetylation and DNA methylation correlate with RGS10 suppression and chemoresistance in ovarian cancer. Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics.